In its broadest aspect, the present invention relates to the conversion of lignocellulosic biomass materials into combustible fuel products. In particular, there is provided a continuous process for fermentatively converting such biomass materials into ethanol using a process design that permits all or part of the process water from the ethanol fermentation process to be recycled so as to significantly reduce the consumption of process water.
Increasing global energy requirements and heightened environmental awareness have resulted in increasing focus on alternatives to fossil fuels as energy sources. Human activity with respect to combustion of fossil fuels contributes significantly to the total amount of carbon dioxide (CO2) released into the atmosphere. Carbon dioxide is purported to be a so called xe2x80x9cgreenhouse gasxe2x80x9d and thus to contribute to global warming.
In contrast to energy production by combustion of fossil fuels, energy production by combustion of contemporary biomass (predominantly in the form of harvested plant material) or fuels derived from such biomass is regarded as being xe2x80x9cCO2-neutralxe2x80x9d, since the amount of CO2 released by combustion of a given amount of such biomass corresponds to the amount of CO2 which was originally taken up from the atmosphere during the build-up of that amount of biomass.
Among fuels derived from plant biomass, ethanol has received particular attention as a potential replacement for or supplement to petroleum-derived liquid hydrocarbon products. To minimise the production cost of ethanol produced from biomass (also referred to in the following as xe2x80x9cbioethanolxe2x80x9d) It is important to use biomass in the form of low-cost by-products from gardening, agriculture, forestry, the timber industry and the like; thus, for example, materials such as straw, maize stems, forestry waste (log slash, bark, small branches, twigs and the like), sawdust and wood-chips are all materials which can be employed to produce bioethanol.
In general, however, the price of bioethanol has not been competitive with that of traditional fossil fuels and it is therefore highly needed to reduce production costs as far as possible by optimising or improving upon bioethanol production technologies.
One important factor in relation to bioethanol production on a commercial scale is the cost of the process water employed. In general, the aqueous effluent from conventional bioethanol production based on the above biomass materials contains substances at a level which, if such process water is recycled, will be rate limiting for the pre-treatment of the lignocellulosic material and/or inhibitory for subsequent hydrolysis of the pre-treated material and fermentation of sugars therein. Accordingly, it is a current practice in bioethanol production to dispose of this water effluent and replace it in the process with fresh process water.
There is thus an industrial need to design bioethanol production processes wherein all or part of the process water can be recycled.
In U.S. Pat. No. 5,221,357 there is described a process for treating a polysaccharide material such as cellulose, hemicellulose and lignocellulose by a two stage acidic hydrolysis to produce monosaccharides and a wet oxidation of the solids such as lignin to produce soluble products e.g. organic acids. The monosaccharides produced are subsequently subjected to fermentation to produce ethanol. Residues from wet oxidation and fermentation are subjected to a methanation step. However, in order to be capable of recycling the remaining liquid and solids into the system a secondary wet oxidation step after methanation is needed which is an additional cost in the production of ethanol.
Thus, the industry is not in the possession of any commercially attractive processes for continuously producing combustible fuel products which permit the process water to be recycled.
It is therefore one significant objective of the present invention to provide a process for continuously processing lignocellulosic material into valuable fuel products wherein the wastewater effluent from the ethanol fermentation effluent is subjected to a treatment, such as an anaerobic fermentation step generating a further combustible fuel product and a wastewater effluent in which the amount of potential inhibitory substances is at a sub-inhibitory level, which in turn permits all or part of the effluent water from the anaerobic fermentation step to be recycled into the process.
The process of the invention thus has the advantages of being capable of 1) giving a very high degree of conversion of carbon in the starting lignocellulosic biomass to useful products, 2) reducing the consumption of water used in the process, and 3) minimising the mounts of residual waste material emerging from the process.
Thus, the process of the invention not only provides improved process economy, e.g. with respect to production of a further combustible fuel product, but is also more environmentally friendly than traditional processes for obtaining such products.
Accordingly, the present invention pertains to a process for continuously converting solid lignocellulosic biomass material into ethanol, the method comprising the steps of:
(i) providing an aqueous slurry of the biomass material,
(ii) subjecting, in a reaction vessel, said aqueous slurry to elevated temperature conditions and/or an oxygen enriched atmosphere to obtain a slurry in which at least partial separation of the biomass material into cellulose, hemicellulose and lignin has occurred,
(iii) subjecting the slurry resulting from step (ii) and/or the aqueous phase hereof to a a treatment resulting in at least partial hydrolysis of the cellulose and hemicelulose to obtain a slurry and/or aqueous phase containing an amount of microbially fermentable sugars that permits the slurry or aqueous phase to be used as an ethanol fermentation medium,
(iv) subjecting the slurry and/or aqueous phase of step (iii) to at least one ethanol fermentation step,
(v) separating the ethanol from the fermentation medium resulting from step (iv) resulting in a fermentation wastewater effluent containing a level of inhibitory substances that, if present in any of the preceding steps (ii) to (iv) would be rate limiting or inhibitory;
(vi) subjecting said wastewater effluent to a treatment whereby the level of the inhibitory substances is reduced to a level that, if the wastewater effluent is introduced into any of the preceding steps (ii) to (iv) is not rate limiting or inhibitory;
(vii) introducing all or part of the thus treated wastewater effluent into any of the preceding steps (i) to (v), and
(viii) continuously repeating steps (i) to (vii).
As shown herein, it was possible to provide a fully operational process for continuously converting solid lignocellulosic biomass material, which process comprises wet oxidation or treatment at an elevated temperature such as steam explosion, enzymatic hydrolysis, ethanol fermentation and finally wastewater treatment. An interesting feature of the process according to the invention is that it is not necessary to incorporate any detoxification steps in the process as all substances produced during each single step of the process served as a substrate for the organisms used in a subsequent step.
As described above, the lignocellulosic biomass material is subjected to a pre-treatment in step (ii), which is wet oxidation or a treatment at an elevated temperature such as e.g. steam explosion. If used, the amount of oxidising agent employed in this step will in general be an amount which is effective to substantially prevent or minimise formation of undesirable reduction products, e.g. furfural and/or furfural derivatives. A well suited oxidising agent is oxygen per se, and presently preferred processes of the invention are performed in the presence of oxygen introduced into the reactor at an initial partial pressure of oxygen equal to or exceeding ambient partial pressure of oxygen.
It appears that cellulose and any hemicellulose present in unsolubilized solid residue which may remain after performing a wet-oxidative or steam explosion treatment in step (ii) is rendered more susceptible relative to cellulose and hemicellulose in lignocellulosic material which has not been treated in the manner of the invention to chemical or enzymatic hydrolysis to give the constituent monosaccharides (D-glucose in the case of cellulose, and primarily D-xylose and/or other pentoses in the case of most hemicelluloses), hereby facilitating procedures such as fermentation to convert glucose or xylose to ethanol or to convert xylose to xylitol or lactose.
In relation to the above-mentioned application of enzymatic treatments or fermentation procedures, use of the process of the invention result in substantial removal of any microorganism- and/or enzyme-inhibitory substances such as acetate, 2-furfural and/or 5-hydroxymethyl-2-furfural, as well as phenolic substances such as vanillin, vanillic acid, homovanillic acid, acetosyringon, syringic acid, syringaldehyde, syringol and the like, which might otherwise accumulate in the process water as a consequence of the degradation of lignin and other substances in the first step of the process, and which may subsequently inhibit microorganisms and/or inhibit the catalytic action of enzymes added for the purpose of facilitating, for example, hydrolysis of cellulose to glucose or hydrolysis of components of solubilized hemicellulose, such as xylans, mannans or arabinans, to the corresponding monosaccharides.
Accordingly, it has now been found that, in order to avoid, in the water used in the process, an accumulation of substances, such as carboxylic acid and other potential fermentation inhibitors produced during the disruption of the structure of lignocellulosic material by means of a pre-treatment such as wet-oxidation or steam explosion and during an ethanol fermentation step, it is possible to remove or at least reduce the amount of these substances to a sub-inhibitory level by applying an aerobic or anaerobic treatment step using one or more microorganisms which alone or together are capable of utilising the carboxylic acids and other fermentation inhibitors as nutrients, the level of which is thereby reduced.
In this manner it is possible to treat the wastewater effluent from the ethanol fermentation process to generate methane or other combustible biogases and a final treated wastewater, wherein the level of inhibitory substances that, if present in any of the steps of the process, i.e. during wet oxidation or steam explosion of the lignocellulosic biomass is performed in order to obtain at least partial separation of said biomass or when present during the subsequently hydrolysis or fermentation of sugars, would be rate limiting or inhibitory for said separation, hydrolysis and/or fermentation. Thus, in the present context, the expression xe2x80x9cinhibitory substancesxe2x80x9d refers to substances such as carboxylic acids which inhibit the pre-treatment of the lignocellulosic biomass material and to substances, such as furans and phenols and carboxylic acids, which inhibit the ethanol fermentation. It appears that a very high percentage (often about 80% or more) of the organic matter, also referred to as chemical oxygen demand (COD) remaining after ethanol fermentation can be converted to biogas, thus minimising the amounts of waste materials emerging from the process.
As mentioned above, step (ii) of the process according to the present invention encompasses a wet oxidation or elevated temperature treatment, e.g. steam explosion of the lignocellulosic material. The terms xe2x80x9cwet oxidationxe2x80x9d and xe2x80x9cwet-oxidativexe2x80x9d as used herein refers to a process which takes place in an aqueous medium, i.e. liquid water or a liquid medium containing at least a substantial proportion of liquid water, in the presence of an oxidising agent which reacts oxidatively in some manner and to some extent with one or more components or species present (as a solid or solids, and/or in dissolved form) in the medium. The process normally takes place at an elevated temperature, i.e. at a temperature significantly above room temperature or normal ambient temperature (usually at a temperature of at least 100xc2x0 C.), and at a pressure at least equal to the vapour pressure of water above the liquid aqueous medium at the temperature in question plus the partial pressure(s) of any other gas or gasses, e.g. oxygen, or (when using air) oxygen plusxe2x80x94primarilyxe2x80x94nitrogen, present. The conditions (temperature, pressure) employed are such that the aqueous medium does not boil. The wet oxidation and the below discussed steam explosion convert a large portion of the biomass material to CO2, H2 O and simpler, more oxidised organic compounds, mainly low-molecular weight carboxylic acids.
As an alternative to wet oxidation the more well known steam explosion (Puls, 1993) or steaming can be successfully used in the process according to the invention. Steam explosion or steaming operate at the same temperature range of 170-220xc2x0 C., e.g. a range of 180 to 210xc2x0 C. and reaction time of 2-20 minutes, but the chemicals used differ and addition of water, prior to the treatment by soaking the biomass in weak acidic or alkaline solutions, is only optional. Steaming operates with saturated steam with or without prior addition of oxygen, carbon dioxide, sulphur dioxide or sulphuric acid as catalyst (Saddler et al, 1993).
As already indicated, processes according to the invention employ lignocellulosic material of plant origin, the lignocellulose, which is the principal component of such materials, in general being built up predominantly of cellulose, hemicellulose and lignin.
Cellulose, which is a xcex2-glucan built up of anhydro D-glucose units, is the main structural component of plant cell walls and normally constitutes about 35-60% by weight (% w/w) of lignocellulosic materials.
Hemicellulose is the term used to denote non-cellulosic polysaccharides associated with cellulose in plant tissues. Hemicellulose frequently constitutes about 20-35% w/w of lignocellulosic materials, and the majority of hemicelluloses consists predominantly of polymers based on pentose (five-carbon) sugar units, such as D-xylose and D-arabinose units, although more minor proportions of hexose (six-carbon) sugar units, such as D-glucose and D-mannose units, are generally also present.
Lignin, which is a complex, cross-linked polymer based on variously substituted p-hydroxyphenylpropane units, generally constitutes about 10-30% w/w of lignocellulosic materials. It is believed that lignin functions as a physical barrier to the direct bioconversion (e.g. by fermenting microorganisms) of cellulose and hemicellulose in lignocellulosic materials which have not been subjected to some kind of pre-treatment process (which may very suitably be a wet-oxidative process as described in relation to the present invention) to disrupt the structure of lignocellulose.
To minimise the production cost of ethanol produced from biomass it is important to use biomass in the form of low-cost by-products from gardening such as garden refuse, waste materials from agriculture, forestry, the timber industry and the like. Thus, processes of the invention are applicable to any kind of hemicellulose-containing lignocellulosic materials. Relevant materials thus include wooden or non-wooden plant material in the form of stem, stalk, shrub, foliage, bark, root, shell, pod, nut, husk, fibre, vine, straw, hay, grass, bamboo or reed, singularly or in a mixture.
Preferred lignocellulosic materials in the context of the invention include wood (both softwood and hardwood), straw, corn stovers and so-called hulls. Wood employed in the context of the invention is generally heartwood (duramen) and/or outer wood (secondary xylem) derived from trunks, stems and/or branches of deciduous or evergreen trees or shrubs. Wood from the roots of such trees or shrubs may also be of value.
Useful sources of wood include numerous species of various genera of coniferous and broad-leaved trees/shrubs. Among conifers may be mentioned the following: Pinaceae, including pines (Pinus spp., such as Pinus sylvestris), silver firs (Abies spp., such as Abies alba), spruces (Picea spp., such as Picea abies), larches (Larix and Pseudolarix spp., such as Larix decidua and L. kaempferi) and Douglas fir (Pseudotsuga menziesii). Among broadleaves may be mentioned the following: Betulaceae, including birches (Betula spp., such as Betula pendufa); and Fagaceae, including beeches (Fagus spp., such as Fagus sylvatica) and oaks (Quercus spp., such as Quercus robur).
Useful sources of straw include in particular cereals (cereal grasses), i.e. gramineous plants which yield edible grain or seed. Straw from, for example, oat (Avena spp., such as A. saliva), barley (Hordeum spp., such as H. vulgare), wheat (Triticum spp., including T. durum), rye (Secal cereale), rice (Oryza spp.), millet (e.g. species of Digitaria, Panicum, Paspalum, Pennisetum or Setana), sorghum (Sorghum spp., including S. bicolor var. durra (also referred to as xe2x80x9cdurraxe2x80x9d) and milo), buckwheat (Fagopyrum spp., such as F. esculentum) and maize (also referred to as corn (Zea mays), including sweetcorn] is well suited for treatment according to the process of the invention.
As employed herein, the term xe2x80x9chullxe2x80x9d generally denotes the outer covering, rind, shell, pod or husk of any fruit or seed, but the term as employed herein also embraces, for example, the outer covering of an ear of maize. Relevant hulls include hulls selected among the following:
hulls from oat (Avena spp., such as A. saliva), barley (Hordeum spp., such as H. vulgare), wheat (Triticum spp., including T. durum), rye (Secal cereale), rice (Oryza spp.), millet (e.g. species of Digiftaa, Panicum, Paspalum, Pennisetum or Setaria), sorghum (Sorghum spp., including S. bicolor var. durra and milo), buckwheat (Fagopyrum spp., such as F. esculentum), maize [also known as corn (Zea mays), including sweetcorn], corn cob, rape-seed (from Brassica spp., such as B. napus, B. napus subsp. rapifera or B. napus subsp. oleifera), cotton-seed (from Gossypium spp., such as G. heraceum), almond (Prunus dulcis, including both sweet and bitter almond) and sunflower seed (Helianthus spp., such as H. annuus).
Hulls of cereals, including not only those mentioned among the above, but also hulls of cereals other than those mentioned among the above, are generally of interest in the context of the invention, and preferred hulls, such as oat hulls and barley hulls, belong to this category. In this connection it may be mentioned by way of example that oat hulls are often available in large quantities at low cost as a by-product of oat-processing procedures for the production of oatmeal, porridge oats, rolled oats and the like; thus, a total of around 75,000 tons of oat hulls is produced per year as a by-product of oat-processing in Denmark, Norway and Sweden together with northern Germany.
Other types of hulls of relevance in relation to processes of the invention include, for example, palm shells, peanut shells, coconut shells, other types of nut shells, and coconut husk.
It should be noted that the native physical form, bulk and/or dimensions of lignocellulosic materials such as wood, straw, hay and the like will generally necessitate, or at least make it desirable, to carry out comminution of the material (e.g. by milling, abrading, grinding, crushing, chopping, chipping or the like) to some extent in order to obtain particles, pieces, fibres, strands, wafers, flakes or the like of material of sufficiently small size and/or sufficiently high surface area to mass ratio to enable degradation of the material to be performed satisfactorily. In the case of wood, material of suitable dimensions will often be available as a waste product in the form of sawdust, wood chips, wood flakes, twigs and the like from sawmills, forestry and other commercial sources.
In contrast, numerous types of hulls, e.g. cereal grain or seed hulls in general, including oat hulls as employed in the working examples reported herein, have in their native form sufficiently small dimensions and a sufficiently high surface area to mass ratio to enable them to be used directly, without prior comminution, as lignocellulosic materials in a process according to the present invention.
The initial ratio of solid lignocellulosic material to liquid aqueous medium in the wet-oxidation reactor will generally be in the range of 0.02-1 kg/liter, often 0.05-0.35 kg/liter, such as 0.05-0.2 kgl/liter, depending on the form, bulk and/or dimensions of the lignocellulosic material as treated. On an industrial scale it will normally be economically most advantageous to perform the process of the invention at the highest practicable ratio of lignocellulosic material to liquid, aqueous medium, i.e. at the highest ratio which permits adequate mixing of the lignocellulosic material in the liquid medium comprising the oxidising agent and which leads to a satisfactorily high rate of degradation of lignocellulose.
By using certain materials of types preferred in the context of the present invention and in the manner disclosed herein it is thus possible, on an industrial scale, to avoid having to use time- and energy-consumingxe2x80x94and thereby expensivexe2x80x94comminution procedures which require investment in, and maintenance of, appropriate comminution apparatus or machinery.
Further to the above, it may nevertheless be desirable with certain types of lignocellulosic materials (e.g. shells of certain nuts) among those of relevance in relation to the present invention to subject the material in question, before treatment by a process of the invention, to a comminution procedure (e.g. by milling, abrading, grinding, crushing, chopping, chipping or the like) in order to enhance the overall reactivity of the material by enhancing, e.g., the physical mobility, mixability, ratio of surface area to mass and the like of the material.
Pre-Treatment of Lignocellulosic Material (Steps (i) and (ii) of the Process According to the Invention)
As described above, the first step in the process for continuously converting solid lignocellulosic biomass material into ethanol, is to provide an aqueous slurry of the lignocellulosic biomass material. The thus obtained slurry is in step (ii) of the process subjected to elevated temperature conditions and/or an oxygen enriched atmosphere to obtain a slurry in which at least partial separation of the biomass material into cellulose, hemicellulose and lignin has occurred.
In one preferred embodiment, the aqueous slurry in step (ii) is subjected to a wet of the oxidation treatment discussed in detail above. In another useful embodiment of the present process, the aqueous slurry in step (ii) is subjected to a steam explosion treatment as also discussed above. In the present context the wet oxidation treatment and the steam explosion treatment of the lignocellulosic biomass material is referred to as pre-treatment. It will be understood that the steam explosion treatment optionally can be performed without providing the lignocellulosic biomass material as an aqueous slurry.
Oxidising Agents
As already indicated, if an oxidising agent is present during the pre-treatment, a preferred oxidising agent in the context of processes according to the invention is oxygen per se.
Other oxidising agents which mayxe2x80x94at suitable concentrations and under suitable conditions of temperature and reaction timexe2x80x94be appropriate for use in a wet-oxidative process in the manner of the invention include, in particular, hydrogen peroxide. Hydrogen peroxide is very soluble in water, is readily available commercially as aqueous solutions of concentration ranging from relatively dilute (e.g. hydrogen peroxide concentrations of around 3% w/w) to relatively concentrated (e.g. hydrogen peroxide concentrations of about 30-35% w/w) and isxe2x80x94like oxygenxe2x80x94a very acceptable oxidising agent from an environmental point of view.
Hydrogen peroxide is thus generally well suited for inclusionxe2x80x94either alone or in combination with one or more other oxidising agents, e.g. oxygenxe2x80x94as an oxidising agent in the liquid, aqueous medium employed, and in such cases the initial concentration of hydrogen peroxide in the liquid, aqueous medium will normally suitably be in the range of 0.5-10% w/w.
Oxidising substances which are not well suited as oxidising agents in the context of the process of the invention include oxidising acids, such as concentrated or dilute nitric acid.
When oxygen is employed as oxidising agent, it is preferredxe2x80x94as mentioned previouslyxe2x80x94that the process is performed in the presence of oxygen introduced at an initial partial pressure of oxygen equal to or exceeding the ambient partial pressure of oxygen (i.e. the partial pressure of oxygen in the surrounding air, which at sea level is normally around 0.2 bar, typically about 0.21 bar), and initial oxygen partial pressures which lie in the range from about 0.2 to about 35 bar will normally be of interest. It is, however, generally prefer-able to employ initial oxygen partial pressures of at least 0.5 bar, normally in the range of 0.5-35 bar. Typical initial partial pressures of oxygen will be in the range of 1-15 bar, such as 3-12 bar, e.g. 5-12 bar. The solubility of oxygen in water at temperatures of relevance for the process of the invention increases with oxygen partial pressure, and the use of such elevated partial pressures of oxygen can thus be advantageous in ensuring the availability of sufficient oxygen in dissolved form.
The oxygen employed may be added in the form of substantially pure oxygen or in the form of an oxygen-containing gas mixture (such as atmospheric air) which in addition to oxygen is constituted by one or more other gases (e.g. nitrogen and/or an inert gas, such as argon) that are not detrimental to the performance of the process of the invention; it will, however, often be advantageous to employ substantially pure oxygen (such as oxygen of xe2x89xa799% purity, which is readily commercially available in conventional gas cylinders under pressure).
When employing oxygen as oxidising agent, an appropriate, effective quantity of oxygen (or oxygen-containing gas mixture) mayxe2x80x94particularly in the case of batch processes in which a chosen quantity (batch) of appropriate lignocellulosic material is treated according to the invention in a reactor which may be closed and, optionally, pressurisedxe2x80x94be introduced into the reactor in question as a single charge at an appropriate initial pressure. Reactors of this type employed in batch processes for wet-oxidative treatment in the manner of the invention will, in addition to containing a certain volume of aqueous liquid phase in which the solid lignocellulosic material in question is contained, generally enclose a free volume or headspace above the liquid phase, and disregarding other considerations it will then be apparent that the greater the ratio of the headspace volume to the liquid phase volume, the lower the initial pressure (partial pressure) of oxygen that will be required to ensure the presence of an effective amount of oxygen gas within the reactor; the partial pressure of oxygen in the reactorxe2x80x94measured at the initial temperature in the reactor or reaction vesselxe2x80x94will decrease during the course of the process of the invention owing to consumption of oxygen in the oxidation reactions which occur.
By way of example only, when a batch reactor which can be closed and pressurised (e.g. a loop-reactor of the type described herein) is operated with an aqueous liquid phase containing about 60 grams of lignocellulosic material per liter of liquid phase, an appropriate effective amount of oxygen will typically be ensured by employing a ratio of headspace volume to liquid phase volume of about 1:1 and an initial oxygen pressure (partial pressure) in the range of 0.2-12 bar. Moreover, since the solubility of oxygen (and a number of other gases, including nitrogen) in water at partial oxygen pressures of interest in the present context increases with temperature above about 100xc2x0 C., and increases rapidly with temperature above about 140xc2x0 C., it will generally be advantageousxe2x80x94not only with such closed batch reactors, but also with other types of reactorsxe2x80x94to employ temperatures in excess of this latter temperature in order to ensure the presence of an adequate concentration of dissolved oxygen; for the same reason it will be possible by increasing the temperature further to employ relatively lower partial pressures of oxygen and still achieve satisfactory concentrations of dissolved oxygen in the liquid, aqueous medium.
As an alternative (which will almost always be employed in the case of continuous or substantially continuous processes, i.e. processes in which lignocellulosic material enters the wet-oxidation reactor essentially continuously, and products of the process exit or are withdrawn from the reactor essentially continuously), oxygen or an oxygen-containing gas mixture may be introduced essentially continuously (or at least at suitably frequent intervals) into the reactor at a suitable pressure so as to ensure the continued availability of sufficient oxidising agent.
Reaction Vessel
Reaction vessels useful to perform the wet-oxidative treatment or steam explosion in step (ii) of the process according to the present invention are usually containers and the like which are generally closed (not open to the surrounding atmosphere) and, optionally, pressurizable reaction vessels; some types of closed, pressurizable reaction vessels suitable for, in particular, batch-type wet-oxidative treatment in the manner of the invention have already been mentioned above. In one embodiment of the present invention, step (ii) is performed as a batch process in a closed, pressurizable reaction vessel having a free volume for containing oxygen-containing gas and/or water vapour.
Relevant types of reaction vessels for performing batch or essentially continuous processes such as wet oxidation or steam explosion include substantially vertically disposed reaction vessels in which the liquid, aqueous medium and the lignocellulosic material in question may be contained and into which oxygen or an oxygen-containing gas mixture (suitably air) may be introducedxe2x80x94continuously or at intervalsxe2x80x94under pressure via one or more inlets, ports. valves or the like situated at or near the bottom of, and for at other locations along the length of, the reaction vessel containing the aqueous slurry of the lignocellulosic material; such reactors, which may suitably, but optionally, have an upper headspace or free volume, may be essentially cylindrical, tubular or of any other appropriate form. Vertical tower reaction vessels suitable for use in the context of the invention are described, for example, in GS 706,686 and GB 812,832.
Reaction vessels for performing continuous or essentially continuous wet-oxidative treatment or treatment at elevated temperatures using e.g. steam explosion in the manner of the invention may, for example, also be tubular or substantially tubular reaction vesselsxe2x80x94very suitably essentially horizontally disposedxe2x80x94through which the liquid phase is pumped or otherwise driven, and which in principle have little or no headspace (free volume) available for, e.g., oxygen in gaseous form. Such reaction vessels will normally comprise one or more appropriately positioned injection inlets, ports, valves or the like for admitting oxygen gas (or, less preferably, an oxygen containing gas mixture) or steam under pressure more or less directly into the liquid phasexe2x80x94e.g. near the beginning of the reaction vessel (reckoned in the direction of flow of liquid within the reaction vessel) and optionally at one or more further positions along the length of the reaction vesselxe2x80x94such that at least a substantial proportion of the introduced oxygen or heated water vapour dissolves in the liquid medium, thereby bringing it into intimate contact with lignocellulosic material in question and thus maximising the oxidising efficiency of the introduced oxygen or the degradation effect of the heated water vapour.
In both batch and continuous wet-oxidative or elevated temperature processes according to the invention, it is generally desirable, where possible, to cause mixing of the aqueous slurry and any gas phase per se which may be present in the reaction vessel. This may suitably be achieved by mechanical stirring of the slurry, although agitation of the reaction vessel as a whole or other means of causing mixing may be applicable. In the case of batch processes employing a recirculatory reaction vessel of the general type as described below (the xe2x80x9cloop-reactorxe2x80x9d in which the liquid phase is recirculated via a tubular section of the reaction vessel by means of a pump, impeller wheel or the like, adequate mixing is generally ensured by the recirculation of the liquid phase (containing lignocellulosic material) at a suitable rate. Thus, one preferred embodiment of the present invention, is where step (ii) is performed as a batch process in a closed, pressurizable reaction vessel with recirculation of the reaction mixture. Similarly, when performing an essentially continuous process in a reaction vessel which is substantially tubular, cylindrical or the like, adequate mixing will often be achieved by causing a sufficiently high rate of flow of liquid phase (containing lignocellulosic material) through the tube(s), cylinder(s) or the like of the reaction vessel.
Temperature
As already mentioned, preferred conditions in step (ii) of the present process include the use of temperatures in the vicinity of, or in excess of, 100xc2x0 C. In general, temperatures in the range of 120-240xc2x0 C., such as 180-220xc2x0 C., more typically in the range of 180-210xc2x0 C., will be appropriate for the vast majority of such embodiments of the process according to the invention, and when using lignocellulosic materials of preferred types it will be usual to employ temperatures in the range of 160-210xc2x0 C., such as 180-210xc2x0 C. Good results appear to be obtainable with temperatures around 185-195xc2x0 C. or 170-190xc2x0 C. As already indicated, the temperature employed should be a temperature at which boiling of the liquid, aqueous medium does not occur under the pressure conditions in question. However, in preferred embodiments, the temperature in which step (ii) is performed is less than 220xc2x0 C., such as less than 200xc2x0 C., e.g. less than 195xc2x0 C. including less than 190xc2x0 C., e.g. less than 185xc2x0 C., such as less than 180xc2x0 C. including less than 175xc2x0 C.
It is, however, desired to set the temperature so as to obtain the desired separation of the lignocellulosic biomass material into cellulose, hemicellulose and lignin, without the destruction of to many polysaccharide molecules, as these molecules serve as a direct nutrient for the ethanol producing organisms in the subsequent step of the present process. As shown in the below Examples, e.g. in Table 2.2, there is a correlation between the reaction time and the temperature used in the reaction vessel. In general it is has been shown that the shorter the reaction time applied the higher temperature is needed in order to obtain a satisfactory separation of the lignocellulosic biomass material.
Heat may be supplied to the reaction mixture (notably the liquid phase/lignocellulosic material) by any suitable method, such as by immersing the reaction vessel in an appropriate heating bath (comprising, e.g., an oil, a molten salt or molten salt mixture, super-heated steam, etc.), by means of thermally conductive (typically metal) tubing which is wound around the outside of the reaction vessel, and for is immersed in the reaction medium itself, and through which suitably hot oil, superheated steam or the like is passed, orxe2x80x94similarlyxe2x80x94by means of one or more electrical resistance heating elements wound around the outside of the reaction vessel and/or immersed in the reaction medium. Other applicable methods of heating include induction heating (e.g. of a metal reaction vessel casing) and microwave heating.
It should be noted here that the degradation reactions taking place in the wet-oxidative treatment or steam explosion treatment which is a preferred feature of the process of the invention normally lead to oxidation or heat effected degradation of a certain proportion of the organic material, notably lignin and some hemicellulose, but also in many cases pectin (which is often present to some extent in lignocellulosic materials), in the lignocellulosic material employed. These oxidative or heat generated reactions are beneficial in the sense that they are, in general, exothermic, and the heat generated thereby contributes to reduce the quantity of thermal energy which has to be supplied to the reaction mixture in the reaction vessel in order to maintain the desired temperature.
Reaction Time
Heating of the lignocellulosic material(s) in the liquid, aqueous medium in a wet-oxidative treatment or by steam explosion in the manner according to the invention will normally be carried out for a period of time ranging from about 1 minute to about 1 hour (i.e. about 1-60 minutes), depending not only on the other reaction conditions (e.g. the reaction temperature, and the type and concentration of oxidising agent) employed, but also on the reactivity (rate of reaction) of the lignocellulosic material. In practicable embodiments of the process of the invention, step (ii) will normally employ reaction times in the range of 5-30 minutes, often 5-15 minutes, and when other reaction conditions are in preferred ranges, such as an oxygen (partial) pressure in the range of about 3-12 bar, e.g. 3-10 bar, and a temperature in the range of about 160-210xc2x0 C., suitable reaction times will often be in the range of about 10 to about 15 minutes.
Adjustment of pH in the Reaction Mixture
In many cases, the treatment performed in step (ii) may be carried out with satisfactory results without any adjustment of the pH, i.e. neutral, of the aqueous slurry before, or during, the performance of the treatment. However, for some types of lignocellulosic materials of relevance in the context of the invention it may be advantageous to adjust the pH of the reaction mixture before and/or during performance of the treatment. The pH may be decreased, i.e. acidic conditions, but in general the pH of the reaction mixture is increased (i.e. alkaline) by adding appropriate amounts of an alkali or base (e.g. an alkali metal hydroxide such as sodium or potassium hydroxide, an alkaline earth metal hydroxide such as calcium hydroxide, an alkali metal carbonate such as sodium or potassium carbonate or another base such as ammonia) and/or a buffer system. Thus, in an interesting embodiment of the present invention the aqueous slurry is subjected to alkaline conditions in step (ii).
As mentioned above, a major objective of the treatment in step (ii) is to break down the lignocellulosic material into hemicellulose and cellulose. Because the dissolved polysaccharides, i.e. cellulose and hemicellulose, and the sugars and carboxylic acids produced during the pre-treatment serve as a direct nutrient source for the microorganisms used in the subsequent ethanol and methane fermentations, respectively, a gentle break down is desired, i.e. the destruction of the polysaccharides is not desired. Thus, an important embodiment of the present process, is wherein at least 60% of the polysaccharide contained in the solid lignocellulosic biomass material is recovered in the slurry and/or aqueous phase after the aqueous slurry has been subjected to a pre-treatment in step (ii), such as at least 60%, e.g. at least 70% including at least 80%, such as at least 90% of the polysaccharides are recovered.
It has been shown that the unsolubilized solid residue remaining after performing step (ii) of the process of the invention appears is well suited for use as animal feed, or as a supplement to animal feed, for animalsxe2x80x94notably ruminants, such as cattle, sheep, goats or deerxe2x80x94of importance in farming or agriculture. The solid residue remaining at this stage, which is generally rich in cellulose fibres, also appears to have applications in the areas of plant-growth media (e.g. in potting soils/composts and in organic media of the peat moss type and the like), soil-improvement agents (materials added to soil to improve, e.g., water retention, soil aeration, root penetration, etc.) and composite materials [structural materials which are produced by combining the solid residue with one or more other materials (e.g. a plastic such as polyethylene or polypropylene) in appropriate ratios, and which have modified properties relative to those of the latter material(s)].
Hydrolysis of the Slurry and/or Aqueous Phase (Step iii of the Process According to the Invention)
Subsequently to the treatment of step (ii) the slurry and/or the aqueous phase hereof is subjected to a treatment resulting in at least partial hydrolysis of the cellulose and hemicellulose to obtain a slurry and/or aqueous phase containing an amount of microbially fermentable sugars that permits the slurry or aqueous phase to be used as an ethanol fermentation medium.
The purpose of such a hydrolysis treatment is to hydrolyse oligosaccharide and possibly polysaccharide species produced during the wet oxidative treatment or steam explosion in step (ii) of cellulose and/or hemicellulose origin to form fermentable sugars (e.g. glucose, xylose and possibly other monosaccharides). Such treatments may be either chemical or enzymatic. However, in accordance with the invention the cellulose may instead of being converted to glucose be used as fibres in the paper industry.
Chemical hydrolysis may normally very suitably be achieved in a known manner by treatment with an acid, such as treatment with dilute (e.g. 2-10% w/w, typically 4-7% w/w) aqueous sulphuric acid, at a temperature in the range of about 100-150xc2x0 C., e.g. around 120xc2x0 C., for a period of 5-15 minutes, such as 5-10 minutes. Treatment with ca. 4% w/w sulphuric acid for 5-10 minutes at ca. 120xc2x0 C. is often very suitable.
Enzymatic hydrolysis may likewise be achieved in a known manner by treatment with one or more appropriate carbohydrase enzymes (glycosidases, EC 3.2). In preferred embodiments, the carbohydrase enzyme is selected from the group consisting of a cellulase (EC 3.2.1.4) in the case of hydrolysis of cellulose or cellulose fragments; a xylanase (such as an endo-1, 4-xcex2-xylanase, EC 3.2.1.8) in the case of hydrolysis of xylans; a xcex2-glucanase including a glucan-1, 3-xcex2glucosidase (exo-1, 3-xcex2 glucanase, EC 3.2.1.58) or an endo-1, 3(4)-xcex2-lucanase, EC 3.2.1.6, in the case of hydrolysis of soluble fragments of cellulose to glucose, a pectinase (polygalacturonase, EC 3.2.1.15) in the case of hydrolysis of pectate and other galacturonans. Commercial enzyme products of relevance in this connection include Celluclast(trademark), available from Novo Nordisk A/S, Bagsvaerd, Denmark, e.g. as Celluclast(trademark) 1.5 L (a liquid preparation). Celluclast exhibits both cellulase activity (degrading cellulose to glucose, cellobiose and higher glucose polymers) and some degree of xylanase activity.
Fermentable sugars, notably monosaccharide product(s), obtained by hydrolysis are useful for further transformation to give other useful products (e.g. ethanol or xylitol). Thus, glucose (derived from cellulose) and xylose (derived from xylans in hemicellulose) may be transformed to ethanol using relevant fermenting microorganisms as described herein, and xylose may, for example, alternatively be transformed to xylitol by established methods (e.g. by catalytic hydrogenation or by fermentation).
Preferred embodiments, include those where the slurry and/or aqueous phase obtained in step (iii) contains, calculated on the total carbohydrate content, at least 40% microbially fermentable sugars, such as at least 50% fermentable sugars, e.g. at least 60% fermentable sugars including at least 70% fermentable sugars.
Ethanol Fermentation (Step iv of the Process According to the Invention)
In a further step of the process according to the invention the slurry and/or aqueous phase of step (iii) is subjected to at least one fermentation step employing one or more fermenting microorganisms capable of degrading oligo- and/or monosaccharides present in said liquid phase to form ethanol.
It will be understood, that it is possible, if desired, to combine process step (iii) and (iv) in the same reaction vessel, and thus performing hydrolysis to microbial fermentable sugars and simultaneously ferment these to ethanol utilising one or more microorganisms.
With regard to fermentation of, e.g., glucose to yield ethanol, any microorganism capable of converting glucose to ethanol can be used in the process according to the invention. For example, a suitable microorganism include a mesophilic microorganism (i.e. one which grows optimally at a temperature in the range of 20-40xc2x0 C.), e.g. a yeast also referred to as xe2x80x9cbaker""s yeastxe2x80x9d, Saccharomyces cerevisiae. 
With regard to fermentation of, e.g. xylose to yield ethanol, any microorganism capable of converting xylose to ethanol can be used in the process according to the invention. Useful microorganisms include e.g. certain types of thermophiles (i.e. organisms which grow optimally at an elevated temperaturexe2x80x94normally a temperature in excess of about 50xc2x0 C.) and genetically engineered microorganisms derived therefrom. In preferred embodiments, a suitable organism for the ethanol fermentation is selected from the group consisting of Thermoanaerobacter species including T. mathranii, Zymomonas species including Z. mobilis and yeast species such as Pichia species. An example of a useful strain of T. mathranii is described in Sonne-Hansen et al., 1993 or Ahring et al. 1996 where said strain is designated as strain A3M4.
It will be appreciated, that a useful ethanol-fermenting organism can be selected from a genetically modified organism of one of the above useful organisms having, relative to the organism from which it is derived, an increased or improved ethanol-fermenting activity. As used herein the expression xe2x80x9cgenetically modified bacteriumxe2x80x9d is used in the conventional meaning of that term i.e. it refers to strains obtained by subjecting a organism to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethanemethane sulphonate (EMS) or N-methyl-Nxe2x80x2-nitro-N-nitroguanidine (NTG), UV light or to spontaneously occurring mutants, including classical mutagenesis. Furthermore, as it is possible to provide the genetically modified bacterium by random mutagenesis or by selection of spontaneously occurring mutants, i.e. without the use of recombinant DNA-technology, it is envisaged that mutants of the above mentioned organism can be provided by such technology including site-directed mutagenesis and PCR techniques and other in vitro or in vivo modifications of specific DNA sequences once such sequences have been identified and isolated.
Using microorganisms with different optimal growth temperature requirements to ferment glucose and xylose, respectively, to yield ethanol, it may thus be desirable to perform the fermentation step in question as a two-stage process wherein the slurry and/or aqueous phase after the preceding step (iii) is first contacted with one of the microorganisms under appropriate conditions therefore (e.g. S. cerevisiae at a temperature of around 30xc2x0 C.) and subsequently with the other microorganism under its appropriate conditions (e.g. T. mathranii at a temperature of about 70xc2x0 C.). The two stages may suitably take place in separate fermentation reaction vessels or in the same reaction vessel in a sequential manner.
Fermentation reaction vessels (fermentors) of any suitable, known type may be employed in performing one or more fermentation steps of the type in question. For further details of suitable reaction vessels, reference may be made, for example, to J. E. Bailey and D. F. Ollis, 1986. Batch fermentation and continuous fermentation are both suited in this connection.
Subsequent to the ethanol fermentation step, the ethanol is separated from the fermentation medium resulting from step (iv) resulting in a fermentation wastewater effluent containing a level of inhibitory substances that, if present in any of the preceding steps (ii) to (iv) would be rate limiting for the at least partial separation of the biomass material and/or he liberation of sugars and ethanol fermentation. As used herein, the expression xe2x80x9cinhibitory substances that, if present in any of the preceding steps (ii) to (iv) would be rate limiting for the at least partial separation of the biomass material and/or the liberation of sugars and ethanol fermentationxe2x80x9d relates to substances produced during the wet oxidation or steam explosion performed in step (ii) and by the ethanol fermenting organisms used in step (iv). Such substances include carboxylic acids such as acetic acid and lactic acid, and furans including 5-hydroxymethylfurfural, 2-furfural and 2-furoic acid and phenols including guaiacol, syringol, 4-hydroxy benzalde-hyde, vanillin, syringaldehyde, 3,4,5-tri-methoxybenzaldehyde, 4-hydroxy aceto-phenone, acetovanillone, acetosyringone, 3,4,5-trimethoxyacetophenone, 4-hydroxy benzoic acid, vanillic acid, syringic acid, p-coumaric acid and ferulic acid.
In addition, the expression xe2x80x9crate limiting levelxe2x80x9d is used in the present context, to indicate a concentration of the above inhibitory substances which inhibits or reduces the performance of the pre-treatment, hydrolysis and/or ethanol fermentation. If the wet oxidation or steam explosion is performed under conditions of increasing concentrations of organic acids, such as carboxylic acids, i.e. when the water used is process water recycled from the process contains a high concentrations of organic acids, the fractionation of the cellulose and hemicellulose is compromised. In addition, more carboxylic acids and furans are produced under the pre-treatment which in a potential concentration inhibits microbial growth.
Treatment of the Wastewater Effluent (Step vi of the Process According to the Invention)
As already indicated, the process according to the invention comprises subsequently subjecting the wastewater effluent obtained in steps (v) and (vi) to a treatment, such as a biological treatment, whereby the level of the inhibitory substances is reduced to a level that, if the wastewater effluent is introduced into any of the preceding steps (ii) to (iv), is not rate limiting for the pre-treatment or inhibiting the hydrolysis and/or ethanol fermentation process.
In a preferred embodiment, such treatment is an anaerobic fermentation process employing one or more anaerobic fermenting microorganisms capable of degrading of converting substances present in said wastewater effluent to form combustible fuel such as methane.
Microorganisms
In one useful embodiment of the present invention, the treatment in step (vi) is performed using methane-producing microorganisms (also known as methanogens) which constitute a unique group of prokaryotes which are capable of forming methane from certain classes of organic substrates, methyl substrates (methanol, methylamine, dimethylamine, trimethylamine, methylmercaptan and dimethylsulfide) or acetate (sometimes termed acetoclastic substrate) under anaerobic conditions.
Methanogens are found within various genera of bacteria, and methanogenic bacteria of relevance in the context of the present invention include species of Methanobacterium, Methanobrevibacter, Methanothermus, Methanococcus, Methanomicrobium, Methane genium, Methanospirillum, Methanoplanus, Methanosphaera, Methanosarcina, Methanolobus, Methanoculleus, Methanothrix, Methanosaeta, Methanopyrus or Methanocorpusculum; some of these, notably species of Methanopyrus, are highly thermophilic and can grow at temperatures in excess of 100xc2x0 C. Only three genera of methanogenic bacteria, viz. Methanosarcina, Methanosaeta and Methanothrix, appear to contain species capable of carrying out the acetoclastic reaction, i.e. conversion of acetate to methane (and carbon dioxide). It will be appreciated that useful methanogenic bacteria can be selected from a genetically modified bacterium of one of the above useful organism having, relative to the organism from which it is derived, an increased or improved methane producing activity. Such a genetically modified organism can be obtained by the methods discussed above.
In the context of the present invention it will generally be most appropriate to apply, in addition to one or more methanogens, other types of microorganisms which, alone or in combination, are capable of degrading organic substances present in the material to be treated in the anaerobic fermentation step of the process of the invention, but which are not directly suited as substrates for the methanogen(s) employed in the anaerobic fermentation step. Such other types of microorganisms include certain fermentative anaerobic bacteria capable of converting, for example, glucose to products such as acetate, propionate, butyrate, hydrogen and CO2, and so-called acetogenic bacteria, which convert organic substances such as propionate, butyrate and ethanol to acetate, formate, hydrogen and CO2.
However, the treatment of the wastewater effluent may also be performed as an aerobic treatment, used aerobic organisms capable of utilising the above mentioned inhibitory substances so as to reduce such substances to a level that, if the wastewater effluent is introduced into the reaction vessel of step (ii) or in any other step of the process, is not rate limiting.
Reaction Vessel Types
The treatment process in step (vi) of the process of the invention is suitably carried out using a reaction vessel of a type known as an xe2x80x9cUpflow Anaerobic Sludge Blanketxe2x80x9d reactor (UASB reactor) as for example described in Schmidt and Ahring (1996). A schematic drawing of a reactor of this type, which normally has the general form of a substantially vertically oriented cylinder, is shown in FIG. 1.
Recycling of the Treated Wastewater Effluent (Steps vii and viii of the Process According to the Invention)
As already indicated above, it is a very important feature of the invention that all or part of the thus treated wastewater effluent remaining after completing the treatment in step (vi) is recycled for reuse as aqueous liquid phase in the process of the invention thereby reducing the consumption of water and minimising the quantity of waste material emerging from the process. By using the treated wastewater effluent for any step of the process according to the invention, i.e. for obtaining the aqueous slurry in step (i) and/or by introducing the treated wastewater effluent into the reaction vessel of step (ii) and/or into the reaction vessel of steps (iii) to (iv), it is possible to continuously repeating steps (i) to (vii), and thus continuously converting solid lignocellulosic material into ethanol and methane.
Accordingly, in preferred embodiments, at least 5% of the wastewater effluent resulting from step (v) is introduced into any step of the process according to the invention, such as at least 10% e.g. at least 20% including at least 30%, such as at least 40% e.g. at least 50% including at least 60% such as at least 70% e.g. at least 80% including at least 90% or even 100%. The introduction of the treated wastewater into the preceding process steps can occur substantially without decreasing the production of ethanol or methane in said steps.
Thus, the purpose of the wastewater treatment in step (vi) of the present process is to reduce the organic matter (COD), i.e. the inhibitory substances, such as carboxylic acids, furans and phenolic compounds, present in the wastewater, in order, when the treated wastewater effluent is reintroduced into the process, to secure that the concentration of inhibitory substances is not at a rate limiting or inhibitory level for the partial separation of the biomass material and/or to the hydrolysis and/or ethanol fermentation. Accordingly, a very high percentage of the organic matter (COD) remaining after ethanol fermentation is converted to biogas. Thus, in preferred embodiments, at least 50% COD remaining after the ethanol fermentation is converted to biogas, such as at least 60%, e.g. at least 70% including at least 80%, such as at least 85%. As shown in the below examples, it is possible by performing step (vi) to reduce the level of the inhibitory substances in the fermentation wastewater effluent present in step (vi) by at least 80%, such as at least 85%, e.g. at the least 90% including at least 95% or even by 100%.